The primary technology in use today for capturing and purifying cells is magnetic beads. In this technology, a suspension of beads is used to treat a sample containing cells. The magnetic beads contain a tag or chemical entity that is selective for cells or for a certain cell type within the sample. After the cells become associated with the magnetic beads, a magnet is used to collect the magnetic beads and captured cells. The magnetic beads may be re-suspended several times with wash solutions to clean the cells. Finally, a solution can be used to release the cells from the beads and a magnet separates the magnetic beads from the cells.
However, magnetic beads can negatively impact cell viability. This problem is mitigated somewhat by the use of magnetic nanoparticles. The magnetic-activated cell sorting (MACS) method available from Miltenyl Biotec utilizes magnetic nanoparticles to isolate cells. Cells expressing particular surface antigens attach to the magnetic nanoparticles.
For example, the isolation of circulating tumor cells (CTCs) from blood is an area of very active interest at present. These cells which are shed into the vasculature from a primary tumor circulate in the bloodstream and constitute seeds for subsequent growth of additional tumors (metastasis) in vital distant organs. CTCs present in the bloodstream of patients with cancer provide a potentially accessible source for detection, characterization, and monitoring of non-hematological cancers.
Magnetic bead methods are slow and do not always produce pure cell populations. In addition, cells isolated on magnetic beads are may not be viable. There exists a need for a column technology that rapidly captures high concentrations of cells, particularly viable cells and then recovers the cells at high purity for research, detection and for other uses.
In the instant invention, it was discovered that living cells can be used as a stationary phase for a new type of liquid chromatography. In this application, analyte reagents flow through a column in which cells attached to the column medium serve as a stationary phase. Analytes interact with the cell-based stationary phase. The extent of interaction of the analytes with the stationary phase can be measured and used. Later, the cell stationary phase may be recovered and analyzed. It is remarkable that in this type of chromatography, both mobile phase analytes and the stationary phase groups can be analyzed and measured. No previously-described chromatography systems have this capability.
Cells may vary in size and can be vulnerable to shear forces and stress which are exerted by a chromatographic device. These forces may be present while the cells are traveling through the chromatographic system including the tubing, frits and column media. The forces may be present while the cells are present on the column media.
Cells may be susceptible to contamination of various sorts including chemical and biological contamination. However a chromatographic device with shielding for cells in the device is unknown. Chromatographic devices cannot restrict or constrict the flow of liquid in any portion of the flow path including the inlet flow into the pump or column or the exit flow from the column. To restrict or constrict flow would make flow of liquid through the column impossible or at the very least, unpredictable, uncertain and uneven. Cells may be subjected to additional shear forces and biological and mechanical stress when the chromatographic device is sealed. In addition to keep cells alive, they must have nutrients, oxygen and removal of waste gas. Sealing a chromatographic system may cause harm to cells, again in unpredictable ways. There exists a need for a chromatographic system that prevents contamination of cells without harming the operation of the chromatographic system or harming the cells.